Stepwise-external calibration has previously been shown to produce sub part-per-million (ppm) mass accuracy for the MALDI-FTICR/MS analyses of peptides up to m/z 2500. The present work extends these results to ions up to m/z 4000. Mass measurement errors for ions of higher mass-to-charge are larger than for ions below m/z 2500 when using conventional chirp excitation to detect ions. Mass accuracy obtained by using stored waveform inverse Fourier transform (SWIFT) excitation was evaluated and compared with chirp excitation. Analysis of measurement errors reveals that SWIFT excitation provides smaller deviations from the calibration equation and better mass accuracy than chirp excitation for a wide mass range and for widely varying ion populations. A ccurate mass measurement has long been recognized as a powerful tool in mass spectrometry, enabling the assignment of unique elemental compositions for small molecules (MW Ͻ 500 Da) [1], and more recently, used for making higher confidence peptide identifications [2]. Accurate mass measurements are carried out using a variety of mass spectrometers. Time-of-flight (TOF) mass spectrometers now provide accuracy within 10 ppm [3,4]. Orbitrap mass measurement accuracies have been reported to be 2 to 5 ppm [5,6]. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry, developed by Comisarow and Marshall [7,8], currently provides the best mass resolution and mass accuracy (Ͻ1 ppm) of all types of mass analyzers [9 -11] and has proven to be useful for protein identification by database searching [2,12]. Mass measurement accuracy (MMA) at the sub part-per-million (ppm) level using internal calibration [13,14] and several ppm using external calibration have been demonstrated [15,16], and these have led to much greater identification specificity, as described in recent reviews [17,18].For FTICR/MS, space-charge is the principal cause of mass measurement error [15,19,20]. The best MMA is obtained by using internal calibration, as this eliminates global space-charge effects [16]. Conventionally, internal calibration is achieved by mixing a calibrant with the analyte. Internal calibration can be achieved without adding calibrant directly into the analyte by using a dual-spray source [14,21] in ESI experiments or by using the internal calibration on adjacent samples (InCAS) calibration method [22,23] in MALDI experiments. However, internal calibration requires having both calibrant and analyte ions present at the same time in the analyzer cell, which congests the mass spectrum and can lead to overlapping peaks. Such issues can be avoided with external calibration, but space-charge shifts of cyclotron frequencies can lead to systematic errors in mass measurement. The most accurate external calibration procedures rely on a calibration equation that accounts for ion intensities [15,16,24], or for matching the ion abundance between the analyte and calibrant spectra, e.g., by automatic gain control (AGC) [14,25]. However, AGC is not applicable to MALDI-FTICR measurements du...